Sumoylation is Largely Dispensable for Normal Growth but Facilitates Heat Tolerance in Yeast

Abstract

Numerous proteins are sumoylated in normally growing yeast and SUMO conjugation levels rise upon exposure to several stress conditions. We observe high levels of sumoylation also during early exponential growth and when nutrient-rich medium is used. However, we find that reduced sumoylation (∼75% less than normal) is remarkably well-tolerated, with no apparent growth defects under nonstress conditions or under osmotic, oxidative, or ethanol stresses. In contrast, strains with reduced activity of Ubc9, the sole SUMO conjugase, are temperature-sensitive, implicating sumoylation in the heat stress response, specifically. Aligned with this, a mild heat shock triggers increased sumoylation which requires functional levels of Ubc9, but likely also depends on decreased desumoylation, since heat shock reduces protein levels of Ulp1, the major SUMO protease. Furthermore, we find that a ubc9 mutant strain with only ∼5% of normal sumoylation levels shows a modest growth defect, has abnormal genomic distribution of RNA polymerase II (RNAPII), and displays a greatly expanded redistribution of RNAPII after heat shock. Together, our data implies that SUMO conjugations are largely dispensable under normal conditions, but a threshold level of Ubc9 activity is needed to maintain transcriptional control and to modulate the redistribution of RNAPII and promote survival when temperatures rise.

1,10-phenanthroline inhibits sumoylation and reveals that yeast SUMO modifications are highly transient

“…This suggests that the toxicity of 1,10-phenanthroline is due to effects of the drug that are not directly or immediately related to reduced SUMO conjugation levels. This is consistent with our recent finding that yeast cells can tolerate a dramatic reduction in sumoylation levels with little to no effect on viability (Moallem et al, 2023 ).…”

“…( B ) Cultures of the Ubc9-AA strain were treated with rapamycin for the indicated durations, then lysates were prepared and analyzed by SUMO and GAPDH immunoblots. Auto-sumoylated Ubc9-AA can be detected in the SUMO immunoblot (“Ubc9-AA-S”; Moallem et al, 2023 ). ( C ) The W303a strain was grown in conditions that permit inhibition of the 26 S proteasome by the drug MG132 (Liu et al, 2007 ).…”

“…To explore the transient nature of SUMO conjugation in a different way, we used the Anchor-away system, using a strain (Ubc9-AA) in which Ubc9 can be rapidly depleted from the nucleus by induced translocation to the cytoplasm upon treatment with rapamycin (Haruki et al, 2008 ; Moallem et al, 2023 ). Although sumoylation levels in the Ubc9-AA strain are constitutively very low (Moallem et al, 2023 ), nuclear depletion of Ubc9 causes a significant further decrease in overall sumoylation in this system, likely because the vast majority of SUMO-conjugated proteins are associated with chromatin, and therefore depend on nuclear Ubc9 for their modification (Baig et al, 2021 ). Strongly consistent with sumoylation being highly transient, we found that treatment with rapamycin for just 5 min was sufficient to eliminate most SUMO conjugations in the Ubc9-AA strain but not in its parental untagged strain (Figs.…”

“…4A ). Previous work showed that heat shock triggers elevated SUMO conjugation levels by increasing sumoylation and reducing desumoylation through partial depletion of Ulp1 (Moallem et al, 2023 ). We investigated whether heat-shock-triggered reduction in Ulp1 levels could offset the loss of SUMO conjugation after treatment with 1,10-phenanthroline.…”

Targeted protein degradation of the CPSF complex by benzoxaboroles through sumoylation

“…Importantly, a similar impact was not seen with cyclohexamide and therefore this transient hypersumoylation is not the direct result of blocking protein synthesis. Interestingly, this is reminiscent of the sumoylation response to heat shock and oxidative stresses in other eukaryotes, where multisubunit protein complexes become hypersumoylated (Moallem et al, 2023, Shao et al, 2023).…”